Estimation of the amount of a substance in a sample, including failure to detect that substance, has utility in a variety of fields. For example, if the substance is a nucleic acid sequence that is diagnostic for a pathogenic organism, it may be useful to know that that particular nucleic acid sequence is undetectable in a sample, or that the quantity of those sequences is decreasing in response to medication. In genetic diagnostics, the estimation of the copy number of a genomic DNA sequence can be diagnostic for the duplication or deletion of a chromosome segment. For example, in fetal ploidy, determination for diagnosis of Downs syndrome can be performed by comparing the copy number of chromosome 21 diagnostic sequences to the copy number of sequences that are diagnostic for another somatic chromosome. Because accurate diagnosis requires distinguishing two copies from three copies of chromosome 21 sequences, accurate quantification of copy number differences is essential. Further examples may be drawn from epidemiology, ecology, or research applications in gene expression, as well as other fields.
In many cases, the substance of interest may be present in the sample in an amount that is too small to be measured directly. Improved methods for nucleic acid amplification signal acquisition and signal analysis, for example for determining small amounts of substances in a sample with higher confidence and/or greater efficiency as compared to existing methods, are needed.